Effects of purification on the bioavailability of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. They are primarily produced by the gram-positive, anaerobic spore-forming bacterium, Clostridium botulinum. In bacterial cultures, secreted BoNTs are associated with non-toxic accessory proteins forming large complexes. Neurotoxin-associated proteins have been shown to play an important role in the oral toxicity of BoNTs by protecting them from degradation and digestion by gastric acid and enzymes. Most toxicity studies using BoNTs have been performed using highly purified toxin. In this study, the toxicities of purified and crude BoNT/A toxin preparations were compared. Protein components secreted into culture supernatants along with BoNT/A were identified by mass spectrometry and the contribution of extra proteins found in the soluble crude toxin extracts to the toxicity of BoNTs was determined in mouse models of oral and parenteral botulinum intoxication. Analysis of crude toxin composition permitted assessment of the impact of accessory proteins on the oral bioavailability of BoNT/A toxin in food matrices.     

Botulinal neurotoxins: revival of an old killer

Botulinal neurotoxins (BoNTs) produced by anaerobic bacteria of the genus Clostridium are the most toxic proteins known, with mouse LD(50) values in the range of 1-5 ng/kg. They are responsible for the pathophysiology of botulism. BoNTs are metalloproteinases that enter peripheral cholinergic nerve terminals, where they cleave one or two of the three core proteins of the neuroexocytosis apparatus and elicit persistent but reversible inhibition of neurotransmitter release. Their specificity of action has made them useful therapeutic agents for many human syndromes caused by hyperactivity of cholinergic nerve terminals. Their range of clinical applications is continuously growing, and BoNT/A is being used extensively as a pharmaco-cosmetic.     

Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.     

Alternative bioassay for the detection of saxitoxin using the speckled cockroach (Nauphoeta cinerea)

Paralytic shellfish poisoning (PSP) toxins produced by cyanobacteria pose a risk to public health as they occur in drinking water reservoirs and recreational lakes and accumulate in the food chain. One of these PSP toxins, saxitoxin (STX) is one of the most toxic nonprotein substances known. Accordingly, there is a requirement to monitor for these toxins. The standard bioassay used to detect these toxins is the mouse bioassay; however, its use is constrained by animal ethics guidelines and practical considerations. Reported here is the use of the globally distributed speckled cockroach Nauphoeta cinerea as a bioassay test organism for the selective detection of PSP toxicity of Anabaena circinalis aqueous extract and STX. N. cinerea was shown to be tolerant to pure cylindrospermopsin (CYN) and microcystin-LR (MC-LR) at doses 10-fold greater than mouse LD?? values while being sensitive to STX. Similarly, N. cinerea was shown to be tolerant of toxin-containing aqueous extracts of Cylindrospermopsis raciborskii, Microcystis aeruginosa, and Nodularia spumigena while being sensitive to A. circinalis. Peak sensitivity to STX was 60 min postinjection with a KD?? of 31.2 ng/g body weight. While this was approximately 3-fold less sensitive than the mouse bioassay, the insect test organism was around 34-fold smaller in mass than a mouse (20 g); thus one-tenth the amount of toxin in absolute quantity was required to reach an ED?? level. The N. cinerea bioassay presents a selective test for PSP toxicity that is rapid, economical, efficient, and simple to perform.     

Comparison of oral toxicological properties of botulinum neurotoxin serotypes A and B

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. Of the seven known serotypes (A-G) of BoNT, serotypes A, B and E cause most of the foodborne intoxications in humans. BoNTs in nature are associated with non-toxic accessory proteins known as neurotoxin-associated proteins (NAPs), forming large complexes that have been shown to play important roles in oral toxicity. Using mouse intraperitoneal and oral models of botulism, we determined the dose response to both BoNT/B holotoxin and complex toxins, and compared the toxicities of BoNT/B and BoNT/A complexes. Although serotype A and B complexes have similar NAP composition, BoNT/B formed larger-sized complexes, and was approximately 90 times more lethal in mouse oral intoxications than BoNT/A complexes. When normalized by mean lethal dose, mice orally treated with high doses of BoNT/B complex showed a delayed time-to-death when compared with mice treated with BoNT/A complex. Furthermore, we determined the effect of various food matrices on oral toxicity of BoNT/A and BoNT/B complexes. BoNT/B complexes showed lower oral bioavailability in liquid egg matrices when compared to BoNT/A complexes. In summary, our studies revealed several factors that can either enhance or reduce the toxicity and oral bioavailability of BoNTs. Dissecting the complexities of the different BoNT serotypes and their roles in foodborne botulism will lead to a better understanding of toxin biology and aid future food risk assessments.     

Detection of paralytic shellfish poisoning (PSP) toxins in shellfish tissue using MIST Alert, a new rapid test, in parallel with the regulatory AOAC mouse bioassay.

In parallel trials with the mouse bioassay, MIST Alert for Paralytic Shellfish Poisoning (PSP), a rapid diagnostic test for PSP, detected 100% of the toxic extracts in over 2100 regulatory samples. Toxic extracts contained at least 80 microg saxitoxin equivalents (STX equiv.) in 100 g of shellfish tissue, or more, as measured by the regulatory AOAC mouse bioassay. Only one potentially toxic sample, which contained 78 and 86 microg STX equiv./100 g shellfish tissue in two different mouse bioassays, was recorded as negative in one replicate of MIST Alert. All other toxic extracts among more than 2100 regulatory shellfish tissue samples were detected by MIST Alert for PSP. The MIST Alert for PSP also detected the majority of extracts containing PSP toxin greater than 32 microg STX equiv./100 g, which is the mouse bioassay detection limit. The MIST Alert for PSP gave a false positive result compared to the mouse bioassay at an average rate of about 14% over all sites, although some differences were seen between sites. Further analysis by high performance liquid chromatography (HPLC) of the (false positive) extracts showed that many contained PSP toxicity in the range of 20-40 microg STX equiv./100 g, below the level detectable by the mouse bioassay. The MIST Alert for PSP gave false positive results from extracts containing less than 20 microg STX equiv./100 g shellfish tissue only about 6% of the time. The PSP family of toxin analogues can occur in any combination in naturally contaminated shellfish tissue and the antibody mixture in the MIST Alert tests detect each of the different PSP toxin analogues with different efficacy. It is therefore impossible to provide an exact detection limit for the MIST Alert that would be applicable for all possible toxin profiles. Through the experience of comparison testing with the regulatory mouse bioassay in many parts of the world, with over 2100 different samples, the MIST Alert for PSP has proven its ability to detect all types of profiles of the PSP toxin analogues. The detection limit for MIST Alert for PSP was about 40 microg STX equiv./100 g for the 'average' profile of PSP toxin analogues. Since the detection limit depends on the toxin profile in the individual extract, it will also vary depending on the profile of analogues most commonly found at each geographic location. This was observed in our study. Over all sites in the trials, approximately 5% of samples below 40 microg STX equiv./100 g were positive, and 5% of samples between 40-80 microg STX equiv./100 g were negative. This is a reflection of the different analogue profiles found in naturally contaminated extracts, even after acid hydrolysis using the AOAC extraction method.     

Avian eyelid assay, a new diagnostic method for detecting botulinum neurotoxin serotypes A, B and E.

Based upon botulinum neurotoxins' (BoNT) mechanism of action, a novel, rapid, and sensitive avian eyelid assay was developed to detect Clostridium botulinum neurotoxin serotypes A, B and E in assay buffer and mimic samples. It showed that chick was the most optimal model of 20-selected laboratory, non-laboratory animals. The eyelid closure of chick was the indicator symptom for positive results. The detection limits achieved range from 5 to 250 mouse LD(50) for toxin types A, B, and E in a buffer system and mimic samples. No cross reactivity occurred when using staphylococcal enterotoxin B, diphtheria toxin and nerve agent sarin, but cross reactivity was obtained in more than 6h for using high dose of tetanus toxin. This cross reactivity can be differentiated by BoNT neutralization tests with a serotype-specific antiserum in parallel. The avian eyelid assay can be performed within as short a time as 0.4-6 h. We report here the development of avian eyelid assay is the second animal bioassay for the detection of toxin types A, B, and E which approaches the sensitivity of the mouse bioassay, and is simple to perform as well as rapid to yield results.     

Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum.

Polycyclic and halogenated aromatic hydrocarbons (PAHs/HAHs) are a diverse group of widespread and persistent environmental contaminants that can cause a variety of detrimental effects in vertebrates. As most available methods to detect these contaminants are expensive, labor and time intensive, and require large amounts of tissue for extraction and analysis, several rapid mechanistically based bioassay systems have been developed to detect these chemicals. Here we describe application and optimization of a recently developed recombinant mouse cell bioassay system that responds to both PAHs and HAHs with the rapid induction of firefly luciferase for the detection of these chemicals in whole serum samples. This chemically activated luciferase expression (CALUX) bioassay has been modified to allow rapid (4-h) and direct analysis of small volumes (25-50 microl) of whole serum in a 96-well microtiter plate format without the need for solvent extraction. This bioassay can detect as little as 10 parts per trillion of the most potent HAH, 2,3,7,8-TCDD, and is also sensitive to other HAHs and PAHs. The use of simple procedures corrects for interplate and intraplate variability and the Ah receptor dependence of the induction response is accounted for by use of the antagonist 4-amino-3-methoxyflavone.     

Rapid Microfluidic Assay for the Detection of Botulinum Neurotoxin in Animal Sera

Potent Botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit foodborne contamination and morbidity. In this report, we describe a rapid 96-well microfluidic double sandwich immunoassay for the sensitive detection of BoNT-A from animal sera. This BoNT microfluidic assay requires only 5 μL of serum, provides results in 75 min using a standard fluorescence microplate reader and generates minimal hazardous waste. The assay has a <30 pg·mL⁻¹ limit of detection (LOD) of BoNT-A from spiked human serum. This sensitive microfluidic BoNT-A assay offers a fast and simplified workflow suitable for the detection of BoNT-A from serum samples of limited volume in most laboratory settings.     

Detection of toxicity of cyanobacteria by Artemia salina bioassay

A bioassay for the detection of neuro- and hepatotoxins of cyanobacteria was developed. Larvae of a small shrimp, Artemia salina, were used as the test organism. Two pure toxins, 44 natural bloom samples, and 29 laboratory strains of cyanobacteria were studied. The toxicity of the samples had earlier been tested by mouse bioassay and in most cases by chemical analysis of the toxins. Moderate and high concentrations of toxins in bloom samples can be reliably detected by the Artemia salina method. Out of 29 toxic bloom samples, 4 were found nontoxic to Artemia. According to mouse bioassay, the toxicity of these samples was very low. The pure laboratory-grown strains contained some compounds that were toxic to A. salina but nontoxic to mouse. Because of these compounds, 1 out of 15 nontoxic bloom samples was erroneously analyzed to be toxic. EC₅₀ for hepatotoxin desmethyl 7-microcystin-RR was 5.0 μ/mL. Pure anatoxin-a-hydrochloride was not toxic to larvae, but when added to nontoxic cyanobacterial samples, anatoxin-a was toxic.     

Diversity of shellfish toxins of "diarrhetic" type revealed by biological and chemical assays.

Extracts of "diarrhetic" shellfish toxins from the edible mussel (Mytilus edulis) were tested with biological and chemical assays to determine toxin content. When tested with the standard mouse bioassay, a higher toxicity was detected in most samples compared to that revealed from detection of the diarrhea-causing substances okadaic acid and dinophysistoxin-1 by high-performance liquid chromatography. Routine extraction of toxins for the two assays was carried out with two different solvents, acetone versus aqueous methanol. Accordingly, we questioned whether the variation in results between the methods could be due to differences in chemical properties of these two solvents. When tested, the two solvent systems showed practically the same efficiency concerning the extraction of okadaic acid and dinophysistoxin-1. This demonstrated that toxins other than those causing diarrhea were present in the samples, and that the mouse bioassay was sensitive to these additional toxins. Subsequent testing of the samples with the mouse bioassay, employing both acetone and methanol extracts, revealed that at least two classes of toxins were present in the mussel samples in addition to okadaic acid and dinophysistoxin-1. It is unclear whether the shellfish toxins revealed in this study are partially from known, nondiarrhetic types, such as pectenotoxins or yessotoxins, or from unknown toxin groups exhibiting ichthyotoxic and hemolytic properties.     

Clinical Uses of Botulinum Neurotoxins: Current Indications,Limitations and Future Developments

Botulinum neurotoxins (BoNTs) cause flaccid paralysis by interfering with vesicle fusion and neurotransmitter release in the neuronal cells. BoNTs are the most widely used therapeutic proteins. BoNT/A was approved by the U.S. FDA to treat strabismus, blepharospam, and hemificial spasm as early as 1989 and then for treatment of cervical dystonia, glabellar facial lines, axillary hyperhidrosis, chronic migraine and for cosmetic use. Due to its high efficacy, longevity of action and satisfactory safety profile, it has been used empirically in a variety of ophthalmological, gastrointestinal, urological, orthopedic, dermatological, secretory, and painful disorders. Currently available BoNT therapies are limited to neuronal indications with the requirement of periodic injections resulting in immune-resistance for some indications. Recent understanding of the structure-function relationship of BoNTs prompted the engineering of novel BoNTs to extend therapeutic interventions in non-neuronal systems and to overcome the immune-resistance issue. Much research still needs to be done to improve and extend the medical uses of BoNTs.     

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay.     

In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics

Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD₅₀ test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1 mouse LD₅₀/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity.     

Repeatability and validity of a fluorimetric HPLC method in the quantification of yessotoxin in blue mussels (Mytilus edulis) related to the mouse bioassay.

Repeatability and validity of a fluorimetric HPLC method in quantification of yessotoxin (YTX) in mussels related to the mouse bioassay was studied. Blue mussels (Mytilus edulis) from the Sognefjord, Norway were sampled from March to November, 1997, and October to December, 1998. A total of 75 samples were analysed for YTX by HPLC using 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3, 4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3, 5-dione (DMEQ-TAD) as a fluorimetric derivatization agent. Among these, 28 of the samples were analysed by HPLC in duplicate. All samples were analysed by the mouse bioassay using both chloroform and ether in the final step of extraction. The duplicate measurements using HPLC was found equal and the method repeatable (p<0.05). The absolute difference between the two measurements was found to increase with increasing level of measurements. This significant positive correlation (p<0.05) was mainly due to concentrations of YTX higher than 200 microg/100g mussel meat. However the precision of the results obtained was not found to be less in the upper level than in the lower level. Based on the internal correlation analysis including the mouse bioassay and the HPLC method a cut-off value of < or =10 microg YTX/5 g digestive gland was found preferable.The mouse bioassay of ether extracts often failed to detect high levels of YTX, and as demonstrated by the low kappa-values, the agreement between the mouse bioassay of ether extracts and the HPLC method was very weak. The HPLC method was found to give repeatable results and thereby found to be reliable. Consequently, the HPLC method seems to the method of choice for detection and quantification of YTX in mussels when compared with the mouse bioassay.     

Production,detection, and quantification of cyanobacterial toxins

Cyanobacterial blooms from several British freshwaters have been toxic by mouse bioassay each year since annual sampling began in 1981. Toxic blooms of Microcystis aeruginosa, Anabaena spp., Gloeotrichia echinulata, Oscillatoria spp., and Aphanizomenon flos-aquae occur, with peptide toxin-producing Microcystis and Anabaena being most often encountered. We are developing a range of detection and quantification methods for cyanobacterial peptide and alkaloid toxins to supplement the standard mouse bioassay. Both types of toxins can be readily assayed by high performance liquid chromatography, and we have developed facile high performance thin layer chromatographic procedures for their detection from natural blooms and laboratory cultures. We have also produced polyclonal and monoclonal antibodies for the assay of Microcystis toxins by enzyme-linked immunosorbent assay and have developed in vitro fibroblast cytotoxicity assays for the toxins of Microcystis and other cyanobacteria.     

Challenges in searching for therapeutics against Botulinum Neurotoxins

Introduction: Botulinum neurotoxins (BoNTs) are the most potent toxins known. BoNTs are responsible for botulism, a deadly neuroparalytic syndrome caused by the inactivation of neurotransmitter release at peripheral nerve terminals. Thanks to their specificity and potency, BoNTs are both considered potential bio-weapons and therapeutics of choice for a variety of medical syndromes. Several variants of BoNTs have been identified with individual biological properties and little antigenic relation. This expands greatly the potential of BoNTs as therapeutics but poses a major safety problem, increasing the need for finding appropriate antidotes.

Areas covered: The authors describe the multi-step molecular mechanism through which BoNTs enter nerve terminals and discuss the many levels at which the toxins can be inhibited. They review the outcomes of the different strategies adopted to limit neurotoxicity and counter intoxication. Potential new targets arising from the last discoveries of the mechanism of action and the approaches to promote neuromuscular junction recovery are also discussed.

Expert opinion: Current drug discovery efforts have mainly focused on BoNT type A and addressed primarily light chain proteolytic activity. Development of pan-BoNT inhibitors acting independently of BoNT immunological properties and targeting a common step of the intoxication process should be encouraged.     

CURRENT METHODOLOGIES FOR DETECTION OF TETRODOTOXIN

In attempt to protect the consumers from TTX-intoxication, the mouse bioassay has historically been the most universally applied tool to determine the toxicity level in monitoring programs. This bioassay, however, shows low precision and requires a continuous supply of a particular size of mice. These potential drawbacks and the world-wide pressure of refraining from the killing of the live animals subsequently led the scientists for developing alternative chemical methods to the mouse bioassay for TTX detection and quantification. Among them, high-performance liquid chromatog-raphy (HPLC) has largely been used in routine work.     

Paralytic shellfish poison (saxitoxin family) bioassays: automated endpoint determination and standardization of the in vitro tissue culture bioassay, and comparison with the standard mouse bioassay.

Mouse neuroblastoma cells swell and eventually lyse upon exposure to veratridine, which, when added together with ouabain, enhances sodium ion influx. In the presence of saxitoxin (STX), which blocks sodium channels, the action of the other two compounds is inhibited and the cells remain morphologically normal. A tissue culture bioassay using mouse neuroblastoma cells, developed by Kogure and colleagues, takes advantage of these principles; in this bioassay, the fraction of the cells protected from the actions of ouabain and veratridine is in direct proportion to the concentration of STX and its analogues. We have modified this bioassay, improving its convenience and speed by eliminating the need to count individual cells to determine the saxitoxin equivalents, and instead have employed a microplate reader for automated determinations of absorbances of crystal violet from stained neuroblastoma cells. When these changes and other minor technical modifications were tested in the tissue culture bioassay systematically, we found the lower detection limit to be around 10 ng STX equivalents (eq) per ml of extract ( = 2.0 micrograms STX eq/100 g shellfish tissue). Our version of the tissue culture bioassay was compared with the standard mouse bioassay using 10 acid extracts of dinoflagellates (Alexandrium excavata and A. fundyense) and 47 AOAC extracts of shellfish tissues. The tissue culture bioassay provided results virtually identical to those obtained with the mouse bioassay (r > 0.96), and moreover, was considerably more sensitive. The results gained from high performance liquid chromatographic (HPLC) analysis of 12 of the same extracts were less consistent when compared with the results from both bioassay methods. The automated tissue culture (neuroblastoma cell) bioassay may be a valid alternative to live animal testing for paralytic shellfish poisoning.     

Strategies to design inhibitors of Clostridium botulinum neurotoxins

Botulinum neurotoxins (BoNTs), produced by spore-forming anaerobic Clostridium botulinum, are the most toxic substances known. They cause the life-threatening disease botulism, characterized by flaccid muscle paralysis. While the natural cases of botulism are rare, due to their extreme toxicity and easy production, BoNTs have become potential biowarfare agents, and create maximum fear among populations concerned with bioterror agents. The only available antidote against BoNTs is equine antitoxin. Equine antitoxin can only target the toxins at extracellular level, and can not reverse the paralysis caused by botulism. In addition, equine antibody can cause severe hypersensitivity reactions, and is limited to be used for prophylaxis treatment. BoNTs are large proteins with three distinct domains, the binding domain, the translocation domain, and the enzymatic domain with highly specific endopeptidase activity to cleave the proteins involved the neurotransmitter release. Targeting any of these domains can inhibit the functions of BoNT. Humanized monoclonal antibodies, small peptides and peptide mimetics, receptor mimics, and small molecules targeting the endopeptidase activity have emerged as potential new inhibitors against BoNTs. With the structure of BoNT resolved, molecular modeling and rational design of potent antidotes against botulism is on the horizon. An area that has not been explored for designing the antidotes against botulism is aptamers, which have been successfully developed as therapeutics in several areas. This review will focus on some of these new strategies to design effective antidotes against botulism. The strategies reviewed in this article can be easily applied to design inhibitors for other bacterial toxins.